The DMA in the chromatin of a eukaryotic cell is highly packed and yet the information contained in the DNA must be made accessible, in a regulated way, to the cellular machinery that decodes it to make the proteins that are the main catalytic and structural components of the cell. The first step in this decoding process, transcription, is carried out by RNA polymerase which functions as a highly processive molecular motor that moves along the DNA template transcribing genetic information from DNA to messenger RNA. During transcription, RNA polymerase and other transcription factors must be able to access the DNA that is packed into nucleosomes and transcription is in part regulated by the accessibility of the DNA. Transcription in chromatin and its regulation must necessarily be highly kinetic processes involving directed molecular motions. Therefore, the long-term goals of our research are to understand on a kinetic and mechanical level (1) the many processes by which the DNA that is packed in nucleosomes is made accessible to RNA polymerase and other transcription factors, (2) how various RNA polymerases transcribe nucleosomal DNA, and (3) the functioning of the regulatory factors involved in these processes. In this project, we propose to use novel and powerful single molecule biophysical approaches that have been recently developed in the Pi's lab to make direct measurements of some of these molecular events. The specific aims of this proposal focus on the effects of chromatin remodeling machines on nucleosomal stability and DNA accessibility, how RNA polymerase overcomes the nucleosome obstacle, and the effects of transcription on nucleosomes. We expect our studies to make significant contributions to the understanding of transcription and its regulation in chromatin.